title: Carcinogenic adducts induce distinct DNA polymerase binding orientations.
abstract: DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA and Department of Medicine, Section of Virology, Imperial College London, London W12 0NN, UK.
authors.forename: Kyle B
authors.initials: KB
authors.lastname: Vrtis
authors.forename: Radoslaw P
authors.initials: RP
authors.lastname: Markiewicz
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Romano
authors.forename: David
authors.initials: D
authors.lastname: Rueda
mesh_terms: D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives* / Q000737:chemistry; D002273:Carcinogens / Q000737:chemistry*; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D018736:DNA Adducts / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D005449:Fluorenes / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods; D011485:Protein Binding
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D005449:Fluorenes; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; C018776:N-(deoxyguanosin-8-yl)acetylaminofluorene; D004247:DNA; D015073:2-Acetylaminofluorene; D004256:DNA Polymerase I; D003849:Deoxyguanosine